This project studies the molecular basis for actin cytoskeleton remodeling in vascular smooth muscle contraction. It complements the other projects of this grants that focus on tissues, cells and macromolecular assemblies. Two families of actin-binding proteins, Ena/VASP and WASP, have emerged as key regulators of cytoskeleton remodeling, playing distinct roles in filament nucleation and elongation, respectively. The molecular mechanisms controlling both processes remain a mystery. Ena/VASP and WASP are functionally distinct but share similar modular structures. They both contain poly-Pro regions that mediate the binding of profilin-actin, followed by G-actin binding domains of the WASP-Homology 2 (WH2) type. This project builds upon important preliminary results (presented here) and our recently determined structures of various WH2- actin complexes to propose the following two hypotheses: 1) Tandem WH2s line up actin subunits along a filament strand forming nuclei for actin assembly (nucleation step). 2) The poly-Pro-WH2 module contributes to filament elongation by "processing" profilin-actin complexes for their incorporation onto the barbed end of growing filaments (elongation step). To test these hypotheses, aims 1 and 2 will dissect the structure- function of the poly-Pro-WH2 and tandem-WH2 modules. We will determine the crystal structures of actin minifilaments assembled via tandem WH2 hybrid constructs and that of poly-Pro-WH2 bound to profilin-actin (transition state in elongation). Complementary solution studies will be carried out using analytical ultracentrifugation and SAXS/WAXS. A biophysical study of the various protein-protein interactions involved in nucleation and elongation will investigate the role of allosteric effects in these processes. Aim 3 studies the actin binding and scaffolding functions of alpha-actinin, a key adaptor protein of the spectrin family. Alpha-actinin mediates the interactions between integrins at the plasma membrane and the focal-adhesion proteins zyxin, vinculin, and paladin, which in turn recruit Ena/VASP to dense plaques. As a paradigm of these interactions, we will study the structural basis for the alpha-actinin-zyxin interaction. Using SAXS/WAXS, FRET and AU, we will test the two prevailing F-actin-binding models, compact and extended, for the actin-binding domain of members of the spectrin family. Understanding the molecular basis of vascular muscle contraction will accelerate the discovery of therapies to treat cardiovascular diseases.